This invention relates to the field of viral constructs, especially their use in gene delivery.
Targeting of a retroviral gene delivery vehicle specifically to the tissue where expression of a transgene is required is a critical aspect of gene therapy. In order to retarget a retrovirus to a tissue of interest, three basic strategies have been widely used to alter retroviral interactions with cellular receptors:
1) Complete substitution of the envelope glycoprotein (Env) by another protein.
2) Incorporation of an antibody fragment into Env.
3) Incorporation of a cell-binding ligand into Env.
Fusing a cell-binding ligand or single-chain antibody fragment to Env only rarely leads to reasonably efficient retargeted gene transfer. The lack of success using these approaches may be due to structural perturbations of the Env molecule as a result of fusion to the novel targeting domain. The present invention reflects an alternative approach to avoid this problem.
In a general aspect, the invention is a retroviral display library, said library comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of retroviruses wherein each retrovirus differs in relation to other retroviruses in the plurality as to the amino acid sequence of an Env protein, each member of the plurality comprising nucleic acid that codes for both said Env protein and a cell-selection marker.
In a general aspect, the invention is a retroviral Env library, said library comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of Env proteins wherein the amino acid sequence of each Env protein differs in relation to the amino acid sequence of the other Env proteins in the plurality.
In another related aspect, the invention is a retroviral nucleic acid library said library comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of retroviral nucleic acid molecules, each of said molecules coding for a retroviral Env protein and a cell-selection marker, and wherein each of said nucleic acid molecule differs in relation to other nucleic acid molecules in the plurality as to the Env protein amino acid sequence that it codes for.
In another related aspect, the invention is a cell population expressing either a retroviral display library or a retroviral Env library or a retroviral Env library of the present invention. Preferably, the population is one that has achieved (or is capable of) such expression through at least 10 (more preferably at least 200) cell population doublings.
In several related aspects, the invention is method. Each of these methods is applicable to any virus. Nevertheless, in one preferred set of embodiments, the virus that forms the basis for the library is one that infects mammalian cells (e.g., human cells). In another preferred set of embodiments, the virus is one of a kind that has been used in gene delivery experiments by others, and includes retroviruses, adenoviruses, herpes viruses, and adeno-associated viruses and alphaviruses. Retroviruses are of particular interest and, in the case of retroviruses, the exterior protein of the virus is preferably an Env protein.
In one aspect, applicable to all viruses, the invention is a method of creating a viral display library said method comprising the steps of:
(1) randomly integrating nucleotides (by adding or, more preferably, substituting nucleotides) into viral nucleic acid molecules, the site of said integration in each nucleic acid molecule being within the coding region for an exterior protein of said virus, so as to create a library of viral nucleic acid molecules; and
(2) infecting a population of cells with the nucleic molecules created in step (1) so as to create a library comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of viruses wherein for each member of the plurality, the amino acid sequence of the exterior protein coded for by the nucleic acid molecule differs from the amino acid sequence of exterior protein coded for by other members of the plurality, and wherein prior to step (2) (preferably prior to step (1)) each of said nucleic acid molecules further comprises a coding sequence for a cell-selection marker.
In another aspect applicable to all viruses, the invention is a method of isolating a virus that can transfer its nucleic acid to a host cell, said method comprising the steps of:
(1) administering to a population of host cells, a random display library of viruses comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of viruses, wherein each virus differs in relation to other viruses of the plurality as to the amino acid sequence of the exterior protein; and
(2) isolating a virus that infected one of said host cells. In one embodiment, each member of the plurality codes, on the same nucleic acid molecule, for both an exterior protein of the virus and a cell-selection marker and step (2) can be achieved by cell selection for virus-infected cells. Alternatively, virus that infected the cells can be identified, after a suitable delay post-infection, in the cell supernatant. For retroviruses, the identification can be made, generally 1 to 2 weeks post infection, by assaying for retroviral reverse transcriptase (Goff, S. P., Traktman, P., and D. Baltimore (1981) J. Virol. 38:239-248).
In another aspect applicable to all viruses, the invention is a method of transmitting non-viral nucleic acid (preferably a gene expressible in said cell., it being understood that the gene can be transmitted as RNA, incorporated into the cell genome as DNA, and be expressed) to a cell, said method comprising the steps of:
(1) administering to a population of host cells, a random display library of viruses, comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of viruses wherein each virus differs in relation to other viruses of the plurality as to the amino acid sequence of the exterior protein,
(2) isolating a virus that infected one of said host cells; and
(3) administering the virus isolated in step (2) to a target cell so as to transfer the nonviral nucleic acid to the host cell, wherein prior to step (3) (preferably prior to step (1)) the nonviral nucleic acid sequence intended for delivery to a host cell is incorporated into a nucleic acid viral molecule of said virus. The target cell may be in an organism (e.g., a human or other mammal), in blood or other tissue outside an organism, or in cell culture (for example, either in liquid suspension or on solid surface). As in steps (1) and (2) of the method of isolating a virus, a cell selection marker may be used.
In another aspect, the invention is a retrovirus, said retrovirus created and isolated by a method of this invention.
A method for screening a viral display library for variants of a virus that target a known tissue-specific surface protein where the library is expressed on the surface of cells, said virus a library virus, and wherein the tissue-specific surface protein is expressed on the surface of a vector virus, said method comprises the steps of:
(1) administering a vector virus to a population of host cells, said vector virus expressing said tissue-specific protein on its surface, said host cells expressing a random library comprising a plurality of exterior proteins of said library virus, wherein the vector virus being administered comprises a nucleic acid molecule coding for a cell-selection marker and wherein the amino acid sequence of each exterior protein in the plurality differs from the amino acid sequence of other exterior proteins of the plurality; and
(2) isolating a cell that bound to the tissue-specific surface protein on the vector virus being administered or isolating a library virus from said cell.
In another aspect applicable to all viruses, the invention is a method for screening a viral display library for viral variants that target a known tissue-specific surface protein where the library is expressed on the surface of cells, said virus a library virus, and the tissue-specific surface protein is expressed on the surface of a vector virus. The method comprises the steps of:
(1) administering a vector virus to a population of host cells, said virus (preferably a pseudotype, such that the tissue-specific surface protein is coded for by a nucleic acid molecule that is not part of the vector virus) expressing said tissue-specific protein on its surface, said host cells expressing a random library comprising a plurality (preferably more than 1xc3x97105, more preferably more than 1xc3x97106) of exterior proteins of said library virus wherein the vector virus being administered comprises a nucleic acid molecule coding for a cell-selection marker and wherein the amino acid sequence of each exterior protein in the plurality differs from the amino acid sequence of other exterior proteins of the plurality; and
(2) isolating a cell that bound to the tissue-specific surface protein on the vector virus being administered or isolating a library virus from said cell. Preferably each cell expresses a small number of amino acid sequence variants of exterior viral proteins, the average number being less than 10, as close to 1 as possible.
For all libraries and all methods, one preferred embodiment is to include a coding region for a nonviral cell-binding peptide (an example is the CCR5-binding peptide discussed herein) in the viral exterior protein of interest, especially within the region that is varied for purposes of constructing the library.
In selecting a virus for delivery of a gene to a cell, it is clearly preferred to select a virus that does not have a pathologic effect on the host cell or host. Therefore, it is preferred to modify a nonhuman retrovirus for use as a vehicle into a human. However, recent advances indicate that lentiviral delivery vectors (such as those derived from the retrovirus HIV-1) have an advantage in that they infect non-dividing cells. Furthermore, Env proteins isolated using the procedures outlined here could be used to eventually pseudotype lentiviral particles or alphavirus particles.
It is preferred that the cell-selection marker, generally a protein, be a drug-resistance marker.
It is understood that a random display library can comprise two or more viruses which have the identical envelope protein amino acid sequence.
The libraries of the present invention, and therefore the related processes of making and using them, are useful as pools of viral vehicles from which an appropriate vehicle can be selected to transfer a gene to a host cell. A large number of possible applications exist for gene therapy vectors created using the present inventions. They can be used to target specific cell types for any gene therapy application. These include the correction of diseases that are caused by enzyme deficiencies such as diabetes. For example, the missing enzyme can be expressed from liver or muscle tissue. Selected Env variants can also be used to target genes to heart cells to deliver factors which promote tissue regeneration in diseased states. Delivery of toxic genes to tumors or virus-infected cells for therapeutic applications can also be accomplished. For lower organisms, such as microorganisms, the methods are applicable for modifying the host range of the viruses for purposes of either killing the microorganism or transferring beneficial genes.